Schizophrenia (SCZ) and autism spectrum disorder (ASD) are severe neurodevelopmental disorders that are caused by the interaction between genetic and environmental risk factors. Large-scale epidemiological have consistently shown that inflammation during pregnancy is associated with an increased risk of SCZ and ASD in offspring. A role for inflammation in the pathogenesis of SCZ and ASD is also supported by animal studies, as well as genetic and post-mortem brain studies. How inflammation during pregnancy contributes to ASD and SCZ at the molecular and cellular level is still largely unknown. The long-term goal of this project is to deepen our insight into the role of inflammatory pathways in ASD and SCZ. To reach this goal we propose to use cerebral organoids derived from human induced pluripotent stem cells (hiPSCs). Cerebral organoid cultures have been shown to closely mimic molecular and cellular processes during the first two trimesters of in utero human brain development. However, protocols up to now were lacking development of microglia, the immune cells of the brain. We have developed an organoid protocol in which microglia develop innately. These organoid-grown microglia resemble primary microglia at the phenotypic and functional level. In addition, we are setting up a novel co-culture model for hiPSC-derived microglia and forebrain organoids. These models provide novel possibilities to study the impact of inflammation on human brain development. The overall objective of this project is to further develop these in vitro models to study the acute and long-lasting cellular changes of the developing human brain induced by maternal inflammation. We will expose microglia- containing organoids to the inflammatory triggers LPS or IFN-? at a developmental stage reflecting the end of the first trimester of gestation in humans. We will harvest the organoids at two early and two later timepoints. In Aim 1. we will characterize the impact of immune activation on microglia number and phenotype, as well as the expression of key inflammatory markers. We will analyze the microglia using immunostainings and the expression of inflammatory markers using qPCR and western blots. Microglia will be sorted and transcriptomic and protein expression changes analyzed using RNA sequencing and CyTOF. In Aim 2. we will investigate the response of other neural cell types to LPS or IFN-? stimulation. We will analyze organoid size and total cell number, as well as the size and number of progenitor zones. In addition, we will analyze the expression of cell- type specific markers in the organoids at different time point after stimulation. On a subsample of organoids, we will perform single cell RNA-seq to determine changes in cell-type composition and cell-type specific changes in gene expression. In the transcriptomic datasets we will analyze whether differential expressed genes are enriched for ASD and SCZ-associated genes. We expect that this project will provide an innovative 3D cell model and novel hypotheses that will be used in follow up studies to deepen our insight into how immune mechanisms contribute to neurodevelopmental disorders.